Progressive multifocal leukoencephalopathy (PML) is a devastating infection of the brain by JC virus, a virus that may remain dormant and inactive until its activation is triggered by immunological disturbances that produce immunosupression in humans. Patients affected by AIDS or immunosuppressive disorders such as cancer or rheumatological disorders are highly susceptible for developing PML. One of the most evident limiting factors in our lack of knowledge of the mechanisms of infection in PML is the lack of reliable in-vitro cell or tissue models for studying the pathobiology of JCV infection. This proposal directly addresses this critical desirable need for the development of an in-vitro brain tissue model of JCV infection. By taking advantage of surgically removed brain tissues from patients undergoing surgical treatment of epilepsy, we developed an in-vitro human organotypic brain tissue culture (HOBTC) system suitable for long-term studies of neuroglial cells, microvascular networks and neuronal-glial interactions. This proposal will further characterize the use of the in-vitro model of JCV infection in HOBTC to assess critical questions about the biology of JCV infection of neuroglia cells and cellular responses to infection, but most importantly to develop a model that may facilitate testing of potential approaches in the treatment of PML. We plan to characterize the profile of neuroglial cell responses to JCV infection in the HOBTC model and also to study the effects of HIV co-infection in the dynamic and patterns of neuroglia infection in the HOBTC model. We will study whether molecular differences and disarrangements in the JCV genome of viruses isolated from CSF of PML patients affect the dynamic and pattern of neuroglia infection in the HOBTC model. Our ability to develop an in-vitro tissue model of PML infection will improve the understanding of the biological mechanisms of JCV infection and above all identify viral and host factors that modulate and determine the pattern of infection of neuroglial cells within the CNS. That ability will also expedite the development of strategies to modify and control JCV infections and to eventually find specific treatments for PML. PUBLIC HEALTH RELEVANCE: Progressive multifocal leukoencephalopathy (PML) is a rare but fatal infection of the brain produced by the JC virus, a virus that may remain dormant in humans until activation produced by immunosupression. The mechanisms and modes of invasion of the brain and remain unknown due to the lack of in-vitro models. This proposal focuses on the development of an in-vitro organotypic brain tissue culture system to study the mechanisms of JCV infection of brain cells and for testing potential strategies for treatment PML.